Throughout this application various publications are referenced by Arabic numerals. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The routine production of human monoclonal antibodies has been of interest since the construction of murine hybridomas which secrete murine monoclonal antibodies of predetermined specificity were originally described (1).
While murine monoclonal antibodies provide valuable tools for the study of biological processes, major limitations are apparent. First, there are restricted number of antigens recognized by these antibodies (50). For example, antibodies directed to polymorphic determinants of the HLA and DR antigens have been difficult to identify (51). Moreover, it has been almost impossible to identify specific human tumor-associated antigens (51-57). Secondly, the pathogenesis of the autoimmune phenomenon in diseases such as diabetes require that the human autoantibodies be defined (58). Finally, therapeutics using murine monoclonal antibodies are restricted due to the formation of antimurine antibodies by the patients receiving the murine monoclonal antibodies for treatment (59-62). It is therefore likely that human monoclonal antibodies will provide major tools for the study of human neoplasia (9-14), autoimmune diseases (2-8), and infectious diseases (16-20), and will serve as potential therapeutic and diagnostic agents for these and other illnesses.
To date, Epstein-Barr Virus (EBV) transformation of antibody-producing human B cells, selection of myeloma serum proteins, and fusion of both murine and human myeloma cell lines or analogs with antibody-producing cells have served as the only practical methods for obtaining human monoclonal antibodies. These methods, however, lack one or more of the features which have made the routine production of murine monoclonal antibodies useful (21-26). While myeloma serum proteins have been used by some investigators as sources of antibodies, this method is dependent upon the large scale screening of rare patients. Lack of reproducibility and continual production, as well as restricted antigen specificities, limit the applicability of this method. EBV virus transformation of antibody-producing B cells has provided the major source of human monoclonal antibodies reported in the literature. There are numerous inherent and methodological problems associated with the use of EBV transformation as a technique for producing antibodies. First and foremost is the instability of monoclonal antibody production by these cell lines (21). Because they have an extremely poor cloning efficiency and unstable antibody secretion, only a few human monoclonal antibody-secreting cell lines have been maintained and have produced sufficient quantities of antibody for use in subsequent studies (3,11). Moreover, the low frequency and lack of specificity of EBV transformation has necessitated selection methods designed to enhance the recovery and transformation of antibody-secreting B cells (21, 29, 30).
The development of human monoclonal antibodies by fusion of myeloma cell lines or analogs with antibody-producing cells has been slowed by two major factors: 1) lack of an appropriate human fusion partner and 2) insufficiently available antigen-specific, human B cells. The presently available human fusion partners are lacking in important characteristics necessary for the production of monoclonal antibodies, i.e., efficient fusion, easy clonability of cell lines and fusion-resulting hybrids, and continuous secretion of large quantities of antibody by the hybrids. Without these characteristics, which are important features of murine fusion partners, it will be extremely difficult to obtain human monoclonal antibodies to many antigens. Human myeloma or lymphoblastoid cell lines have been used for fusion, but frequently these have either a low fusion efficiency, poor growth and cloning, or unstable secretion by the resulting hybrids (6, 23, 31-33). For example, NSI, a murine myeloma cell line, fuses with an efficiency of 1/10,000 with mouse spleen cells (66). Comparative fusion efficiency of LiCron HMY-2, SK007, UC729-6 or GM 1500 is between 1/500,000 and 1/1,000,000 with human cells (6, 33, 67, 68). In addition, several of these cell lines, including derivatives of UC729-6 and LTR228, fuse poorly with normal peripheral blood mononuclear cells (PBM). High fusion efficiency is particularly important in a human system because of the relative rarity of antibody-producing B cells, even in individuals undergoing programmed immunization. In optimally tetanus immunized volunteers, as few as 1 out of 10,000 circulating B cells secrete anti-tetanus antibody (38). Since B cells represent less than 10% of circulating PBM, large numbers would be needed to obtain a single antibody-secreting hybrid. Direct comparisons of a number of human myeloma cell lines, mouse myeloma cell lines, and human lymphoblastoid cell lines as human fusion partners have generally indicated fusion efficiencies on the order of 1/10.sup.5 -10.sup.6 cells, with poor stability, and secretion between 100 ng and 10 .mu.gm/ml in routine cultures (6, 23, 31).
As an alternative to presently available human and murine cell lines used as fusion partners, a number of investigators have attempted to construct myeloma Analogs that might be superior for human monoclonal antibody production. Murine hybridization experiments have shown that fusions between B cells with undifferentiated characteristics and B cells with more differentiated characteristics result in the promotion of those differentiated characteristics in the hybrids (43, 44). Thus, Laskow, et al., and others, were able to promote the appearance of phenotypic characteristics of a more differentiated B cell, specifically including intact immunoglobulin production or secretion, by fusing undifferentiated B cells with a myeloma cell line (43-46). In the attempts to-construct a human myeloma analog that would retain the desirable characteristics in the human fusion partner, it was theorized that the appropriate selection of cells for hybridization would result in the sequential improvement of a series of constructed myeloma analogs (25, 26). These human myeloma analogs were constructed by the fusion of a non-secreting human myeloma cell line with a variety of human cells at selected stages of differentiation. In these studies, while fusion efficiency was high and growth characteristics were excellent, stable secretion of monoclonal immunoglobulin was obtained only from fusions with established malignant human cell lines already committed to secretion. Antibody secretion was rapidly lost by the cloned hybridomas. It is possible that the choice of the non-secreting human myeloma cell line as the basis for the series of constructed human myeloma analogs may have had an impact on the ability of subsequently generated human myeloma analogs and hybridomas to support stable antibody production.
As an alternative to analogs formed by the fusion of human myeloma cells with human cells, heterohybridomas have been constructed by the fusion of murine myeloma cells with human cells (34-37). Some investigators, including the present inventor, have constructed human-mouse myeloma analogs by fusing murine myelomas with a variety of human cells. The murine myelomas used for fusion derive principally from the MOPC21 cell line, developed by Potter and associates and adapted to in vitro growth by Horibata and Harris (27, 28, 39). This cell line and derivatives thereof are routinely used in the production of murine monoclonal antibodies as the fusion partner. Teng, et al., fused MOPC21 with the human cell line SK007 (34), Ostberg and Pursch fused it with a human B lymphocyte (37), and Foung, et al. fused normal peripheral blood lymphocytes with a derivative of SP2, a murine myeloma hybrid (35). Carroll, et al., compared a number of these human-Rouse myeloma analogs for fusion efficiency, immunoglobulin secretion and stability (36). A heterohybridoma, K6H6/B5, constructed by fusion with NSI and human B lymphoma cells, was found to be superior to the other human-mouse myeloma analogs. This heterohybridoma has a fusion efficiency of 1/10.sup.5 cells with 60% of the hybridomas secreting immunoglobulins. Immunoglobulin secretion by the hybrids was on the order of 2-3 .mu.gm/ml.
Despite this effort, most heterohybridoma analogs have proven to have unstable secretion (25, 26) or a poor fusion efficiency when compared to murine myeloma cell lines. Since specific human antibody-producing cells are rare in the peripheral blood, a higher fusion efficiency is a desirable feature of a human fusion partner (38).
The present invention provides a new human-mouse myelomas analog, which has been termed HMMA 2.11TG/0, a method oil constructing it and a method of routinely using the human-mouse myeloma analog for the production of human monoclonal antibodies. The HMMA 2.11TG/0 cell line has an extremely high fusion efficiency with normal PBM and EBV transformed PBM. It clones readily and, once cloned, stably secretes large amounts of human monoclonal antibody.
Human Immunodeficiency Virus 1 (HIV-1) infection represents a new and extremely serious health threat. The evolving epidemic has spread to numerous risk groups in this country and new related viruses have now appeared in Africa and other geographical areas (69-73). Patients with HIV-1 infection may develop a variety of directly related illnesses, including frank Acquired Immunodeficiency Syndrome (AIDS), AIDS Related Complex (ARC), encephalopathy, and AIDS related malignancies. These complications of HIV-1 infection are thought to derive in large part from a progressive and profound immunosuppression which occurs during the course of the illness, or to associated, possible, direct effects of the virus on specific organs, such as the brain (69-73). At the present time it is not known which patients infected with HIV-1 will go on to manifest increasingly serious and morbid complications of the disease, and why some, but not all, individuals will undergo this progression. It is felt that a profound depression of cellular immunity, as a manifestation of viral mediated destruction of T cells, may be involved in this process. Inversion of the normal T4/T8 ratio, depletion of lymphocytes bearing the CD4 antigen, and lymphopenia in infected individuals are strongly correlated with progression to AIDS (74, 75). The preferential infection of T4 lymphocytes, syncytia formation and death of infected T4 cells, and the ability of some antibodies directed at the CD4 complex to prevent infection of cells by HIV-1, have implicated this population of T cells and the CD4 complex in the pathogenesis of this disease (72, 76-79).
While studies of immunodepression in this disease have concentrated on the cellular arm of the immune response, the humoral immune system has also been profoundly effected by HIV-1 infection. Patients infected with HIV-1 have diminished responses to immunization with potent immunogens such as Keyhole Limpet Hemocyanin and Hepatitis B vaccines (80-82). Paradoxically patients also have serum hypergammaglobulinemia, possibly as a result of chronic, non-specific, B cell stimulation. Several pieces of evidence support this contention. Isolated B cells from infected individuals are more likely to spontaneously secrete immunoglobulins as well as specific antibodies, including antibodies to HIV-1 (80-82). These circulating B cells also appear activated on the basis of cell surface phenotypic changes (83). In addition, circulating B cells from HIV-1 infected patients are less likely to be transformed by exogenously added Epstein Barr Virus (EBV), although spontaneous outgrowth of EBV transformed B cells is higher than that seen in normals (82). Because EBV transformation preferentially occurs in non-activated B cells, these data support the notion that circulating B cells are chronically activated (84). Taken together, these studies demonstrate that alteration of the humoral immune response is a major occurrence in HIV-1 infection.
The importance of the humoral immune response to HIV-1 in the in vivo control of the disease is controversial. Some epidemiologic studies of risk groups have suggested that the presence of serum antibody reactive with the gp120 envelope protein of the HIV-1 virus, and capable of neutralizing virus in infection assays, is correlated with lack of disease progression (85-87). Longitudinal studies of thalassemic patients, infected via frequent blood transfusion, and of patients infected during treatment for curable malignancies support the notion that neutralizing antibodies play a role in preventing the development of AIDS (85, 87). Conflicting evidence has also been presented to suggest that these antibodies may have little role in preventing Kaposi's Sarcoma, a manifestation of HIV-1 infection (88, 89). At least one report has suggested that serum antibodies capable of blocking reverse transcriptase activity are also correlated with continued lack of disease progression (90). While the presence of neutralizing antibodies, and antibodies inhibiting reverse transcriptase activity, may be important in the in vivo control of this disease, they may also represent epiphenomena of HIV-1 infection and their presence or absence may be unrelated to the direct cause of further immunosuppression. This might pertain if, for example, viral infection occurred via cell-cell interactions (91). Alternatively, genetic variation or drift in viral envelope proteins may lead to escape from humoral immune control, although some epidemiologic studies have suggested that epitopes involved in viral binding and neutralization are frequently conserved across isolates (92-95). A third mechanism for escape from control might involve dysregulation of the humoral immune system leading to down regulation of antibody synthesis through the destruction of the CD4 lymphocyte population (74, 82). It might be speculated that this process could involve production of anti-idiotypic antibodies some of which may bind to T cells and contribute further to immunosuppression (96, 97). Thus, despite the tentative, speculative, and conflicting data regarding the humoral immune response to HIV-1, it is of great importance that the relationship of the antibody response to HIV-1 and progression of the disease be understood since there remains a real likelihood that this response may significantly alter the course of infection.
Murine monoclonal antibodies reactive with either the CD4 complex or HIV-1 envelope proteins are capable of inhibiting infectivity of HIV-1 in in vitro systems (77-79). These monoclonal antibodies are being used to study idiotypic responses, CD4 attachment, and antigen binding sites on HIV-1 related proteins. For example, a murine monoclonal anti-idiotypic antibody, but not polyclonal rabbit anti-idiotypes, reactive with CD4 binding murine monoclonals reacts with HIV-1 envelope proteins and inhibits cellular infection (79).
Despite the obvious utility of many of these antibodies and their availability for study, the precise determinants involved in the human humoral immune response remain unknown. In order to study both the human humoral response to HIV-1 infection, and the regulation of this response, it is important to obtain the human equivalent of the currently available murine monoclonal antibodies. Human monoclonal antibodies to HIV-1 provide a series of uniform reagents that would be useful in determining the precise epitopes involved in the immune response to this virus, the idiotypic restrictions in the humoral immune response, and the potentially important impact of anti-idiotypic regulation. Moreover, human monoclonal antibodies serve as useful diagnostic and therapeutic reagents in the evaluation and treatment of the disease. Therapeutic advantages of human monoclonal antibodies over murine monoclonal antibodies include a decreased potential for direct immunization against the antibodies (98). In addition, anti-idiotypic antibodies binding to populations of normal T cells prove useful in studying the human immune response in general (99).